The gram-negative bacterium Caulobacter elaborates a paracrystalline protein surface layer (S-layer) which covers the surface of its outer membrane (Smit, et al., 1981; 1992). The S-layer protein monomer is secreted by a Type I secretion mechanism relying upon a C-terminal secretion signal which remains attached to the rest of the protein during the secretion process (Gilchrist, et al., 1992; Bingle, et al. 1997a; and, Awram and Smit 1998). Once the protein monomer is secreted, the S-layer forms by a process of self-assembly as a hexagonal array of about 40,000 interlinked protein monomers (Nomellini, et al., 1997). Anchoring of the S-layer protein to the cell surface is dependent on a smooth lipopolysaccharide (LPS) molecule and Ca+2 ions (Walker, et al, 1994) and involves the N-terminal portion of the S-layer monomer (Bingle, et al, 1997 a,b). The abundance, cell-surface location and geometrical packing of the S-layer protein as well as the inherent properties of Caulobacter (ease of genetic manipulation, simple growth requirements, non-pathogenic nature, and biofilm-forming characteristics) have led to the exploitation of the Caulobacter/S-layer system for biotechnology development (Smit, et al., 2000).
Through use of gene fusions encoding the C-terminal secretion signal of the S-layer monomer linked to sequences encoding a heterologous peptide, the Caulobacter/S-layer system is capable of secreting large quantities of the heterologous peptide (PCT patent applications published under No. WO 97/34000 and WO 00/49153; and, Bingle, et al., 2000). Such Caulobacter expression systems are available commercially under the trade name PurePro™ (Invitrogen, Carlsbad, Calif.). The PureProm™ system is designed for expression of “C-terminal hybrid proteins” in which a heterologous peptide is linked to the S-layer monomer C-terminal secretion signal and does not form an S-layer on the host.
The Caulobacter/S-layer system is also used for expression of heterologous peptides inserted into sites within a full-length (or nearly full-length) S-layer monomer resulting in production of what is termed herein “full-length hybrid protein”. In such cases, a sufficient portion of the N-terminal region of the S-layer monomer is present to provide anchoring of the hybrid protein to the cell surface. The C-terminal secretion signal is present to permit secretion of the hybrid protein. Various preferred sites for insertion of heterologous peptides into the S-layer protein have been reported (WO 97/34000; and, Bingle, et al., 1997(b)). The hybrid S-layer protein so produced is capable of self-assembly on the cell surface resulting in an array of hybrid S-layer protein monomers forming as an S-layer on the cell surface. In this manner, heterologous peptides may be expressed and presented on the cell surface. This technology has particular use in expression and presentation of antigens.
A phenomenon observed during the development of the Caulobacter/S-layer system was the apparent proteolytic cleavage of various hybrid proteins. This was initially observed for both C-terminal hybrid proteins and full-length hybrid proteins. No obvious site specificity was associated with this phenomenon despite monitoring the degradation of numerous modified S-layer proteins comprising different heterologous peptides of varying lengths inserted at different places in the S-layer protein monomer (Bingle, et al. 1997a,b) it has now been reported (Simion, B. et al. 2001) that smaller proteins seen contaminating preparations of C-terminal hybrid proteins are not the result of proteolytic activity but rather the result of internal translation initiation following Met residues within the heterologous or “passenger” portion of the hybrid protein. Nevertheless, the cleavage phenomenon still places limitations on the use of Caulobacter as an expression system for full length hybrid proteins, particularly when the “passenger” peptide is unknown or uncharacterized, since it would then be difficult to know if this phenomenon has affected the hybrid product.
The use of a biological system to express and display a panel or library of different peptides to assess the ability of the peptides to bind to a chosen target, has become a powerful tool for investigating interaction of cellular components (see U.S. Pat. Nos. 5,223,409 & 5,571,698). In this methodology, nucleic acids each encoding a different peptide plus a signal for display of the peptide on the outer surface of a biological system, are introduced into the system. The peptides are expressed and binding domains in the peptides are displayed on the outer surface of the biological system. The system is exposed to target molecules and those members of the system which bind target molecules are isolated and the nucleic acids amplified. Successful binding domains are then characterized. This general method of exposing a variety of peptides, each displaying a different putative binding region, is termed “panning” herein. To date, phage is the preferred biological system for use in panning methodologies. This is partly due to difficulties in the use of bacterial systems for expression and display of heterologous peptides.